Mechanistic details of a protein-protein association pathway revealed by paramagnetic relaxation enhancement titration measurements

Abstract

Protein-protein association generally proceeds via the intermediary of a transient, lowly populated, encounter complex ensemble. The mechanism whereby the interacting molecules in this ensemble locate their final stereospecific structure is poorly understood. Further, a fundamental question is whether the encounter complex ensemble is an effectively homogeneous population of nonspecific complexes or whether it comprises a set of distinct structural and thermodynamic states. Here we use intermolecular paramagnetic relaxation enhancement (PRE), a technique that is exquisitely sensitive to lowly populated states in the fast exchange regime, to characterize the mechanistic details of the transient encounter complex interactions between the N-terminal domain of Enzyme I (EIN) and the histidine-containing phosphocarrier protein (HPr), two major bacterial signaling proteins. Experiments were conducted at an ionic strength of 150 mM NaCl to eliminate any spurious nonspecific associations not relevant under physiological conditions. By monitoring the dependence of the intermolecular transverse PRE (Gamma(2)) rates measured on (15)N-labeled EIN on the concentration of paramagnetically labeled HPr, two distinct types of encounter complex configurations along the association pathway are identified and dissected. The first class, which is in equilibrium with and sterically occluded by the specific complex, probably involves rigid body rotations and small translations near or at the active site. In contrast, the second class of encounter complex configurations can coexist with the specific complex to form a ternary complex ensemble, which may help EIN compete with other HPr binding partners in vivo by increasing the effective local concentration of HPr even when the active site of EIN is occupied.

Fig. 1.

Intermolecular PRE profiles observed on U-[²H,¹⁵N]-labeled EIN from a paramagnetic EDTA-Mn²⁺ moiety on HPr E5C (Top), E25C (Middle), or E32C (Bottom) at an ionic strength of 150 mM NaCl. The concentrations of EIN and HPr are 300 and 450 μM, respectively. Theoretical intermolecular PREs () back-calculated (15) from the coordinates of the specific EIN/HPr complex (28) are shown as black lines. Experimental intermolecular PREs are displayed as filled-in circles as follows. The nonspecific intermolecular PREs (i.e., PREs attributed to the encounter complex ensemble) are in purple and defined by the following criteria: and (because a large value of precludes the reliable identification of a superimposed nonspecific PRE). The specific intermolecular PREs (i.e., PREs attributed to the specific complex) are in black and defined by the criteria that both and (see Methods). The remaining intermolecular PREs are in gray. Crosses indicate residues with ¹H-¹⁵N cross-peaks that are broadened beyond detection by PRE. (Inset) Specific (black) and nonspecific (purple) intermolecular PREs mapped onto the surface of EIN (gray ribbons) with HPr (yellow) in the specific configuration; the EDTA-Mn²⁺ label is represented by a three-conformer ensemble (15) with the Mn²⁺ atoms shown as orange spheres.

Fig. 2.

Summed and normalized intermolecular PREs observed on U-[²H,¹⁵N]-labeled EIN upon titration with paramagnetically labeled HPr-E5C (yellow), HPr-E25C (orange), and HPr-E32C (green). Intermolecular PRE data arising from the specific complex and nonspecific encounter complex ensemble are shown on the left and right, respectively. Data points were normalized to the highest value of each titration. A simple one-site binding curve with K _D = 7 μM is displayed as a black line in both panels. A linear fit to the E5C data is shown as a dotted line on the right.

Fig. 3.

Examples of HPr titration curves for the three classes of intermolecular PREs arising from the EIN/HPr encounter complex ensemble. (A) Class I, exemplified by the HPr-E5C titration curve for Glu74 of EIN, scales linearly with the concentration of the specific EIN/HPr complex (blue line). (B) Class II, exemplified by the HPr-E5C titration curve for Ala161, scales linearly with the concentration of free HPr in solution (red line). (C) Class III, exemplified by the HPr-E5C titration curve for Gly66, behaves as a mixture of classes I and II and fits to a scaled sum (purple line) of the specific EIN/HPr (blue dashed line) and free HPr (red dashed line) concentrations (see Materials and Methods for calculation details). (Inset) Cartoon representations of (A) class I and (B) class II encounter complex configurations of HPr (yellow) on the surface of EIN (gray), with the binding site on each protein shown in green.

Fig. 4.

Bar graphs displaying intermolecular PRE rates observed on EIN originating from the two classes of EIN/HPr encounter complex ensembles. The data shown are for the final point of the HPr titration with 300 μM of EIN and 450 μM of HPr-E5C (Top), HPr-E25C (Middle), or HPr-E32C (Bottom). Relative contributions of the nonspecific PREs that scale linearly with either the concentration of the specific EIN/HPr complex (class I) or the concentration of free HPr (class II) are shown in blue and red, respectively. Intermolecular rates back-calculated from the structure of the specific EIN/HPr complex (15) are displayed as a black line, and nonspecific PREs that are too large (> 65 s^-1) to measure accurately are shown as pink bars.

Fig. 5.

Intermolecular PREs at the final titration point mapped onto the surface of EIN. Displayed as gray ribbon models in the top row and surface representations in the bottom two rows, EIN (light gray) is color coded according to the predominant (> 80% contribution from classes I or II) type of intermolecular nonspecific PRE observed at a particular residue: blue, red, and purple indicate nonspecific PREs with rates that scale linearly with the concentration of the specific EIN/HPr complex (class I), the concentration of free HPr (class II), and a mixture of both (class III), respectively. Nonspecific PREs that are too large to measure accurately (Γ₂ > 65 s^-1) are in pink, and specific PREs with Γ₂ values > 25 s^-1 are in dark gray. Purple and red ellipses, labeled Patch 1 and Patch 2, respectively, highlight two regions on the surface of EIN where the class II nonspecific PREs cluster. In all three rows, HPr-E5C, HPr-E25C, and HPr-E32C (yellow ribbons) are shown in the specific configuration, and the paramagnetic EDTA-Mn²⁺ tags are displayed as a three-conformer ensemble with the Mn²⁺ atoms depicted as spheres (orange).

Fig. 6.

Equilibrium binding model for the EIN/HPr association pathway. The intermolecular PRE data presented here indicate that, upon collision, EIN and HPr form two distinct types of weak, transient encounter complex ensembles: interactions that are in competitive equilibrium with the specific complex (Bottom Left), and interactions that can form simultaneously with the specific configuration to create a HPr_nonspecific/EIN/HPr ternary complex ensemble (Bottom Right). At the concentrations present in our experimental conditions, this second type of encounter complex rarely occurs in the absence of a specifically bound HPr and, therefore, the binary complex involving the second type of encounter complex interactions is displayed as partially transparent (Bottom Center).